Bu çalışma NIH hibe EY019312 ve EY021126 (VJK), Washington Üniversitesi Göz Hastalıkları Anabilim Dalı ve Görsel Bilimler EY002687 tarafından desteklenen ve Araştırma tarafından Körlük Önlemek için.

Tüm deneysel protokoller Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu ile uyumlu olduğunu ve Washington Üniversitesi&#39;nde kurumsal Hayvan Çalışmaları Komitesi tarafından kabul edildi. 1. Perfüzyon ve Numune Tutucu Kurma <ol><li> Deney gününde taze retina perfüzyonu için çözelti hazırlayın. Distile ve deiyonize su kullanın. Aşağıdaki üç çözümlerden birini kullanın. <ol><li> , Medya ve NaHCO 3 1.9 g &#39;Ames 1 şişe (1 L) çözeltisi&#39; Ames bikarbonat içeren hazırlanması </li><li> (MM olarak) Locke çözeltisi hazırlayın: 112.5 NaCl, 3.6 KCl, 2.4 MgCl2, 1.2 CaCl2, 10.0 HEPES, 20.0 NaHCO 3, 3.0 Na süksinat, 0.5 Na glutamat, 0.02 EDTA ve 10.0 glikoz,% 0.1 MEM vitamin ve amino asitler </li><li> (MM olarak) Hazırlama HEPES-tamponlu Ringer solüsyonu: 133.3 NaCl, 3.3 KCl, 2.0 MgCl2, 1.0 CaCl2, 10.0 glikoz, 0.01 EDTA, 12.0 HEPES, pH1 M NaOH 5,8 mi ~ ile 7.5&#39;e ayarlandı, 0.72 g / L kültür ortamı Leibovitz L-15 ekleyin. Herhangi bir perfüzyon medya ile fotoreseptör yanıtı izole etmek için 100 mcM BaCl 2 - 50 mcM DL-AP4 ve 50 - 20 kullanın. </li></ol></li><li> (MM olarak) elektrot 28 için çözelti hazırlayın: 140.0 NaCl, 3.6 KCl, CaCl2, 3 HEPES, 0.01 EDTA 2.4 MgCl2, 1.2 ve 7.4 pH ayarlamak - NaOH ile 7.5 olarak değiştirilmişti. Elektrot çözeltisi bir kaç ay için oda sıcaklığında muhafaza edilebilir. </li><li> Hazırlayın ve numune tutucu test edin. <ol><li> Numune sahibinin taban kısmının kubbelerin üstünde siyah / gri filtre kağıdı Tutkal (Şekil 1A bakınız). Kubbelerin düz üstleri kenarlarda dikkatlice iki bileşenli 5 dk epoksi tutkal sürün. Gerekirse, diseksiyon mikroskobu altında bunu. </li><li> Tutkal neredeyse kuru (yaklaşık 4 dakika) kadar bekleyin ve düz öğesini kullanarak kubbe üzerinde filtre kağıdı bastırın. Tutkal filtre kağıdı deneyden önce en az bir gün.Filtre kağıdı, birden çok kez kullanılabilir ancak kayıtların bir ay sonra değiştirilmelidir. Yedek kağıt yüklemeden önce herhangi bir yapışkan kalıntısı dikkatlice kubbeleri temizlemek için% 70 etanol kullanın. </li><li> Elektrot çözelti hava kabarcıklarını önlemek ve elektrot kanallarına dişli adaptör ile kapalı pelet elektrotlar (Şekil 1A ve B) vida çalışıyor elektrot kanalları doldurun. Dört vida ile numune tutucu üst ve alt parçaları bağlayın ve elektrot çözeltisi ile perfüzyon çizgileri doldurmak. </li><li> Multimetre (Şekil 1B) kullanarak her bir elektrot çiftinin uçları arasındaki direnci ve voltajı ölçün. Direnç 100 Ωk ve 10 mV altında geriliminin kanalları kabarcık-ücretsiz ve elektrotlar iyi şartlarda iseniz altında olmalıdır. </li></ol></li><li> Cam şişe perfüzyon medya 700 ml - 400 dökün. Başka bir 300 mi retina ve s diseksiyon kullanılacak AyrıBir buzdolabına yırttı. Eşanjör bloğunda perfüzyon tüp ayarlayın ve ısıtıcı plaka üzerinde ısıtılmış blok yerleştirin (Şekil 1D bakınız). </li><li> (Şekil 1D bakınız) (Ames &#39;ya da Locke kullanılması durumunda) bağlantıları 2 / O 2 gazı CO olan bir kapakla şişe ve perfüzyon tüp için sarmalar. 37 o C medya ile şişeyi Onceden ve ısıtma plakası veya 37 ayarlanmış su banyosunda hafif stimülasyon birimi üzerine yerleştirin - 39 o C 17. <ol><li> Prime yerçekimi ile çalışan akmayı başlatmak için perfüzyon ortamı ile doldurularak perfüzyon hattı. <ol><li> LKC sisteminde, daha sonra deney sırasında şişede perfüzyon çözeltisi düşürücü seviyesi etkilenmeden akış hızı regülatörü ile ayarlanır önemli bir yer çekimsel bir akış sağlamak için stimülatör ünitesi 17 üzerine şişe yerleştirin. </li><li> Ocuscience sisteminde, s yernumune tutucu uzun perfüzyon çıkış hatları gürültüyü en aza indirmek ve yerleştirmek amacıyla Faraday kafesi içinde erfusion şişe çözümünün yerçekimi sürücü artırmak için de numune tutucu (ve perfüzyon şişesinin) seviyesinin altına (adım 2.8 bakınız). Bu çıkış hatlarını koruyun ve ERG sinyal elektromanyetik gürültü bağlanmasını engellemek için amplifikatörün toprağa kalkanı bağlayın. </li></ol></li></ol></li><li> Debi regülatörleri kullanarak / dk 5 ml - 3 perfüzyon ayarlayın. Doğru regülatör ile bir silindirden% 5 CO 2 /% 95 O 2 gazı bağlayın ve bir hava taşı ile şişe medyanın sürekli köpüren sağlamak için akış hızını ayarlayın. </li></ol> 2. Numune Hazırlama <ol><li> Düz kanatlı microscissors, bir ya da iki 45 o cımbız, jilet ve filtre kağıdı dikdörtgen bir parça da dahil olmak üzere, temiz ve keskin diseksiyon aletleri birleştirin. </li><li> Soğuk perfu yaklaşık 200 ml dökünbüyük bir Petri kabındaki yon çözeltisi (kubbe dahil olmak üzere), numune tutucu bütün alt kısmı çözeltisi içine daldırılmış şekilde. Kubbelerin üzerindeki retina monte ederken Bu adım önemli hale (adım 2.6 bakınız). Bazı çözeltiler karbojen (% 5 CO 2/95% O x 2), doymuş üzere tasarlanmış olmasına rağmen, bu diseksiyon amaçları için gerekli değildir ve burada tarif edilen deneylerde yapılmadı. </li><li> Kayıtların önce 12 saat - Tipik bir deney için, aydınlık / karanlık döngüsü ve 6 onları koyu adapte 12/12 saat içinde hayvanlar tutmak. (Mikroskop ışık kaynağının önünde, örneğin kırmızı filtre kullanın) loş kırmızı ışık altında servikal dislokasyon tarafından takip CO 2 inhalasyon yoluyla hayvan Euthanize ve loş kırmızı veya kızılötesi ışık altında aşağıdaki işlemleri yapmak. <ol><li> Cımbız kullanarak gözleri dışarı çekin ve medya koyun. Kağıt (örneğin, bazı düzenli filtre kağıdı) küçük bir parça üzerinde bir seferde bir göz yerleştirin ve olarak yapmakCımbız ile göz (bu burada çözümün dışında yapılır) tutarken ora serrata düzeyinde yaklaşık yaktı. </li></ol></li><li> Ora serrata boyunca kesin (ya da yakın gözün ekvatora) microscissors ve kornea ve lensi çıkarın. Büyük Petri kabındaki soğuk medyada göz fincan koyun ve diğer gözle aynı işlemi tekrarlayın. </li><li> Mümkün olduğunca sağlam olarak retina tutmak için retina ve sklera arasındaki makas tutarak optik sinir doğru göz fincan üstünden küçük bir kesi kesin. Kavrama iki cımbız kullanarak ve retina ayırmak için birbirinden uzakta cımbız çekin tarafından kesi her iki taraftan sklera. <ol><li> Optik siniri kesin ve uzak yüzeye fiziksel temas minimum miktarda retina izole etmeye çalışın. RPE çoğunlukla diseksiyon işlemi sırasında retina otomatik olarak ayırmak olacaktır. Bu gerçekleştirmek için daha retinanın mekanik rahatsızlık önlemek için daha önemlidirDiseksiyon hızlı bir şekilde, genel olarak retina yanıtı özellikleri üzerinde önemli etki olmadan çözelti içinde en az 30 dakika inkübe edilebilir. </li></ol></li><li> Numune tutucu (Şekil 1C) kubbe üzerinde retina monte edin. Disseke retina ile Petri kabındaki numune tutucu alt kısmını daldırın. Kubbenin üzerindeki retinayı yukarı fotoreseptör tarafı (izole retinanın dışbükey yüzeyi), Slayt ve retina filtre kağıdı üzerinde verdiği, böylece numune tutucu kaldırın. Diğer retina için aynı işlemi tekrarlayın. </li><li> Elektrik retina arasındaki karışma yanı sıra gürültü ve sinyal şant önlemek için dikkatli bir şekilde tutucu plakayı kurutun. Numune tutucu tutucu alt ve üst parçaları arasındaki çözüm dökülme önlemek için yanı sıra bireysel retina fotoreseptör ve ganglion hücre tarafı elektrik izolasyonu yardım etmek için alt kısmı kubbe etrafında O-halkalarını vardır. (Dört vida ile sahibinin üst kısmını takınŞekil 1B) ve bir şırınga ve iğne kullanılarak perfüzyon çözeltisi ile perfüzyon kanalları doldurun. </li><li> Isı değiştirici blok sonraki numune tutucu aktarın ve numune tutucu (Şekil 1D) giriş ve çıkış perfüzyon hatlarını bağlayın. (Üst elektrotlar amplifikatör toprak / eksi pin bağlantı) ERG amplifikatör elektrotlar bağlayın ve bir adaptör kullanılarak numune tutucuya stimülatörü / kontrol ünitesi takın veya bağlı stimülatörü ünitesine ısıtma yastığı kaydırın in vivo hangi ERG sistemi kullanılmaktadır. </li></ol> 3. Kayıtlar <ol><li> In vivo sistemin yazılımını kullanarak amplifikatör ve uyarıcı ayarlarını yapılandırın. 300 Hz filtreleme 1 ile 10 kHz ve low-pass arasında bir değere satın alma frekansını ayarlayın. Yüksek geçiren filtreleme kullanmayın. 60 Hz (veya Avrupa&#39;da 50 Hz) Gerekiyorsa filtreyi çentik kullanın. </li><li> Işık uyarımı ve zekâ olmadan Tutanak temelh loş stimülasyon (örn., -35 dB veya 0.3 mCd şiddetindeki sm -2 yeşil ışık) elektrotlar ve retina örnek arasında iyi elektrik bağlantısı için test etmek. Retina sıcaklık ve fonksiyon istikrarlı duruma ulaşması ve böylece veri toplama başlamadan önce 20 dakika - 10 bekleyin. <ol><li> Herhangi bir özel deneye göre uyaran parametrelerini seçin ve veri toplama başlar. Örneğin, 0 dB kadar ya da 0.1 1.000 kadar mCd şiddetindeki sm den -40 yeşil ışık kullanmak -2 bir skotopik yanıt aile için. Çubuklar arka plan ışığı tarafından bastırılmış gereken Fotopik kayıtlarda yaklaşık 2 parlak log-birimleri 3 yanıp söner kullanın (3-30 Cdm -2) ya da bir prob flaş (-2 = 1 dB yaklaşık 1.3 Cd sm, bakınız Şekil 3). </li><li> Ancak, tam ışık şiddeti değerleri sistem kalibrasyonu ve deneysel koşullara biraz bağımlı olabileceğini unutmayın. Genel bir kural olarak, aşağı 5 ölçek yoğunlukları - 10 kat in vivo karşılaştırıldığında </em&gt; kayıtlar yeşil ışık 17 kullanarak. (Ticari bağdaştırıcı sistemine dahil) retina üzerinde delikler olan bir siyah kapağı numune tutucu ışık saçılması: azaltmak ve in vivo sistemler ticari Ganzfeld çevrede her iki retina homojen ve eşit bir uyarım kolaylaştırmak için kullanılabilir. NOT: koyu adapte retina kayıtları, tipik bir deney ağartıcı pigment sadece önemsiz bir kısmını kullanılan test yanıp söner yoğunlukları böylece RPE odaklı rejenerasyon eksikliği söz konusu değildir. Buna ek olarak, daha önce koni pigment rejenerasyon hala Müller hücresi görsel döngüsü 20 üzerinden izole retinada yer alabilir gösterilmiştir. </li></ol></li><li> Deneyler birkaç saat 30 dakika kadar tipik son gibi bu değişiklikler teknik sorunlara işaret edebileceğiniz gibi, deney sırasında bazal sürüklenme, gürültü seviyesi ve yanıt istikrarı izlemek. NOT: Artan gürültü ya da azalmad tepki genlikleri hava perfüzyon veya elektrot kanalları kabarcıklar, çok düşük ya da yüksek sıcaklık, çözüm sızıntı / dökülme veya retina deplasman gösterebilir. </li></ol> 4. Temizlik <ol><li> Perfüzyon hatları numune tutucu ayırın açın ve filtre kağıdı retina yıkayın. Elektrotları çıkarın ve damıtılmış su (etanol kullanmayın) ile durulayın. Etanol ve / veya distile su ile (perfüzyon kanalları dahil) numune tutucu temizleyin. Dikkatlice yıkayın perfüzyon boru 70&gt; ile% etanol. </li><li> Damıtılmış su (deterjanlar kullanmayın) ile perfüzyon şişesini durulayın. Etanol, aynı zamanda şişe temizlemek için de kullanılabilir. </li></ol>

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Sci. 47, 2583-2588 (2006).</li><li>Walter, P., Luke, C., Sickel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotics+and+light+responses+in+superfused+bovine+retina.">Antibiotics and light responses in superfused bovine retina.</a> Cell. Mol. Neurobiol. 19, 87-92 (1999).</li><li>Luke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+safety+profile+of+alkylphosphocholines+in+the+model+of+the+isolated+perfused+vertebrate+retina.">The safety profile of alkylphosphocholines in the model of the isolated perfused vertebrate retina.</a> Graefes Arch. Clin. Exp. Ophthalmol. 248, 511-518 (2010).</li><li>Januschowski, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+toxicity+testing+of+VEGF+Trap-Eye+in+an+isolated+perfused+vertebrate+retina+organ+culture+model.">Electrophysiological toxicity testing of VEGF Trap-Eye in an isolated perfused vertebrate retina organ culture model.</a> Acta Ophthalmol. 92, e305-e311 (2014).</li><li>Kolesnikov, A. V., Kefalov, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transretinal+ERG+recordings+from+mouse+retina:+rod+and+cone+photoresponses.">Transretinal ERG recordings from mouse retina: rod and cone photoresponses.</a> J Vis Exp. , (2012).</li><li>Koskelainen, A., Hemila, S., Donner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+sensitivities+of+short-+and+long-wavelength+sensitive+cone+mechanisms+in+the+frog+retina.">Spectral sensitivities of short- and long-wavelength sensitive cone mechanisms in the frog retina.</a> Acta Physiol. Scand. 152, 115-124 (1994).</li><li>Lyubarsky, A. L., Falsini, B., Pennesi, M. E., Valentini, P., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV-+and+midwave-sensitive+cone-driven+retinal+responses+of+the+mouse:+a+possible+phenotype+for+coexpression+of+cone+photopigments.">UV- and midwave-sensitive cone-driven retinal responses of the mouse: a possible phenotype for coexpression of cone photopigments.</a> J. Neurosci. 19, 442-455 (1999).</li><li>Lyubarsky, A. L., Daniele, L. L., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+candelas+to+photoisomerizations+in+the+mouse+eye+by+rhodopsin+bleaching+in+situ+and+the+light-rearing+dependence+of+the+major+components+of+the+mouse+ERG.">From candelas to photoisomerizations in the mouse eye by rhodopsin bleaching in situ and the light-rearing dependence of the major components of the mouse ERG.</a> Vision Res. 44, 3235-3251 (2004).</li><li>Azevedo, A. W., Rieke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+protocols+alter+phototransduction:+the+implications+for+retinal+processing+at+visual+threshold.">Experimental protocols alter phototransduction: the implications for retinal processing at visual threshold.</a> J. Neurosci. 31, 3670-3682 (2011).</li><li>Carter-Dawson, L. D., LaVail, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rods+and+cones+in+the+mouse+retina.+I.+Structural+analysis+using+light+and+electron+microscopy.">Rods and cones in the mouse retina. I. Structural analysis using light and electron microscopy.</a> J. Comp. Neurol. 188, 245-262 (1979).</li><li>Fain, G. L., Matthews, H. R., Cornwall, M. C., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+in+vertebrate+photoreceptors.">Adaptation in vertebrate photoreceptors.</a> Physiol. Rev. 81, 117-151 (2001).</li><li>Calvert, P. D., Strissel, K. J., Schiesser, W. E., Pugh, E. N., Arshavsky, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-driven+translocation+of+signaling+proteins+in+vertebrate+photoreceptors.">Light-driven translocation of signaling proteins in vertebrate photoreceptors.</a> Trends Cell Biol. 16, 560-568 (2006).</li><li>Schneeweis, D. M., Schnapf, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+photovoltage+of+macaque+cone+photoreceptors:+adaptation,+noise,+and+kinetics.">The photovoltage of macaque cone photoreceptors: adaptation, noise, and kinetics.</a> J. Neurosci. 19, 1203-1216 (1999).</li><li>Heikkinen, H., Vinberg, F., Nymark, S., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesopic+background+lights+enhance+dark-adapted+cone+ERG+flash+responses+in+the+intact+mouse+retina:+a+possible+role+for+gap+junctional+decoupling.">Mesopic background lights enhance dark-adapted cone ERG flash responses in the intact mouse retina: a possible role for gap junctional decoupling.</a> J. Neurophysiol. 105, 2309-2318 (2011).</li><li>Gouras, P., MacKay, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+in+amplitude+of+the+human+cone+electroretinogram+with+light+adaptation.">Growth in amplitude of the human cone electroretinogram with light adaptation.</a> Invest. Ophthalmol. Vis. Sci. 30, 625-630 (1989).</li><li>Peachey, N. S., Goto, Y., al-Ubaidi, M. R., Naash, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+mouse+cone-mediated+electroretinogram+during+light+adaptation.">Properties of the mouse cone-mediated electroretinogram during light adaptation.</a> Neurosci. Lett. 162, 9-11 (1993).</li></ol>

St Louis Washington Üniversitesi Xenotec, Inc. ile bir lisans anlaşması olan ve ex vivo adaptörün satışından elde bir telif alabilirsiniz.

<html> Biz burada bir ex vivo ERG adaptörü ile birlikte in vivo ERG sistem bileşenlerini kullanarak iki izole fare retina aynı anda yüksek kaliteli ex vivo ERG kayıtları elde etmek için kritik adımlar göstermektedir. Bu çalışmada, aynı solüsyonu (ya Ames &#39;, Locke ya da Ringer) ile hayvandan her iki retina perfüze ama uyuşturucu test amaçlı, farklı bir çözüm örneğin her retina serpmek de mümkündür. yüksek kaliteli veri elde etmek için en öneml...

Elektroretinogram (ERG) ışıkla tetiklenir retina elektriksel aktivitesi kaydetmek için kullanılabilen bir köklü bir tekniktir. ERG sinyal retinanın rezistif dışı uzayda akan radyal akımların neden olduğu gerilim değişiklikleri (fotoreseptör ve bipolar hücrelerin ekseni boyunca) ağırlıklı olarak üretilir. İlk ERG sinyali bir balık gözü 1 yüzeyinden Holmgren, 1865 kaydedilmiştir. Einthoven ve Jolly 1908 2 b-, a- denilen üç farklı dalgaların içine ışık başlangıcından ERG yanıtı, ve c-dalgalar, şimdi bipolar hücreler ON fotoreseptör esas aktivitesini yansıttığı bilinmektedir ve pigment epiteli bölünmüş Hücreler sırasıyla 3-8. ERG, yerel (mikroelektrotla (ex vivo) izole sağlam retina arasında, izole edilmiş bir göz Hazırlama 9&#39;da elde edilen, anestezi uygulanmış hayvanlar ya da (in vivo) insan gözüyle 3,10-15 ya arasında belirli bir retina katmanları kaydedilebilirERG) 4,16. Bunlardan in vivo ERG şu anda retina fonksiyonunu değerlendirmek için en yaygın kullanılan yöntemdir. Bu teşhis amaçlı kullanılabilir veya hayvanlarda veya hastalarda retina hastalıklarının ilerlemesini takip etmek invaziv olmayan bir tekniktir. Ancak, in vivo ERG kayıtları genellikle ekstraoküler fizyolojik gürültü (örneğin, solunum ve kalp aktivitesi) ile kirlenmiş, birkaç örtüşen bileşenleri ile karmaşık bir sinyal üretmek. Yerel ERG retina spesifik katmanlar arasında sinyalini kaydetmek için kullanılabilir, ancak, en invaziv ve diğer ERG kayıt konfigürasyonları ile karşılaştırıldığında düşük bir sinyal-gürültü oranı (SNR) sahip olmasıdır. Yerel ERG ayrıca teknik olarak ve pahalı donanımları (örn mikroskop ve mikromanipülatörler) gerektirir. Sağlam, izole retina gelen Transretinal ERG (ex vivo ERG) istikrarlı ve HIG izin in vivo ve yerel ERG yöntemleri arasında bir uzlaşma sunuyorhayvanlar ya da insanlar 17 sağlam retina h SNR kayıtları. Son zamanlarda, bu yöntem, memeli primat ve insan retina 18-20 çubuk ve koni fotoreseptör fonksiyonunu incelemek için başarıyla kullanılmaktadır. Buna ek olarak, ex vivo olarak, retina pigment epiteli olmaması, ERG sinyalinin pozitif Cı dalga bileşeni çıkarılır ve önemli bir negatif yavaş PIII bileşeni ex vivo kayıtları ortaya çıkar. Yavaş PIII bileşeni retina 21-23 Müller glia hücrelerinin aktivitesi kaynaklı gösterilmiştir. Bu nedenle, ex vivo olarak ERG yöntemi de sağlam retinada Müller hücreleri incelemek için kullanılabilir. Çeşitli çalışmalarda da bu ex vivo ERG kayıtları retina 24 civarında farmakolojik ajanların konsantrasyonu ölçmek ve uyuşturucu 25-27 güvenliğini ve etkinliğini test etmek için kullanılan olabilir göstermiştir. In vivo sistemlerde birden fazla ticari mevcuttur vemutlaka geniş elektrofizyoloji altyapıya sahip olmayan birçok laboratuvarlarında kullanılan. Buna karşılık, ex vivo cihazlar son zamanlarda 17 kadar ve sadece çok az laboratuarlar anda bu güçlü tekniği yararlanarak bir sonucu olarak mevcut olmamıştır. Bu retina fizyolojisi ve patolojisi hakkında bilgimizi ilerletmek ve hastalıkları kör için yeni tedavilerin geliştirilmesi için daha fazla laboratuvarlara ex vivo ERG kayıtları kullanılabilir hale getirmek için yararlı olacaktır. Burada, bir basit ve uygun fiyatlı bir ex vivo ERG aygıtı 17 göstermektedir ve bu çubuk ve koni aracılı sinyal kaydetmek için çeşitli ticari olarak temin edilebilir, in vivo ERG sistemleri ile kombinasyon halinde kullanılabilir göstermek (a- ve b-dalgası) ve fonksiyonu görmemiş vahşi tip fare retinaları Müller hücrelerinin (PIII yavaş).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>In vivo ERG system</td>
			<td>OcuScience</td>
			<td>HMsERG</td>
			<td><a href="http://www.ocuscience.us/id77.html">www.ocuscience.us/id77.html</a></td>
		</tr>
		<tr>
			<td>In vivo ERG system</td>
			<td>LKC Technologies</td>
			<td>UTAS-E 3000</td>
			<td><a href="http://www.lkc.com/products/UTAS/bigshot.html">www.lkc.com/products/UTAS/bigshot.html</a></td>
		</tr>
		<tr>
			<td>Ex vivo adapter</td>
			<td>OcuScience</td>
			<td>Ex VIVO ERG adapter</td>
			<td><a href="http://www.ocuscience.us/id107.html">www.ocuscience.us/id107.html</a></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>North Central Instruments</td>
			<td>Leica M80</td>
			<td><a href="http://www.ncimicro.com/store/ListProduct.asp?bSub=T&amp;idCategory=27">May use any brand</a></td>
		</tr>
		<tr>
			<td>IR emitter</td>
			<td>Opto Diode Corp.</td>
			<td>OD-50L</td>
			<td><a href="http://www.optodiode.com/">www.optodiode.com</a></td>
		</tr>
		<tr>
			<td>Prowler Night Vision Scopes</td>
			<td>B.E. Meyers Electro Optics</td>
			<td>D4300-I</td>
			<td>Military grade product.</td>
		</tr>
		<tr>
			<td>Red filter</td>
			<td>Rosco Laboratories</td>
			<td>Roscolux #27 Medium Red</td>
			<td>May be used instead of IR system</td>
		</tr>
		<tr>
			<td>Red head light</td>
			<td>OcuScience</td>
			<td>ERGX011</td>
			<td><a href="http://www.ocuscience.us/catalog/i29.html">www.ocuscience.us/catalog/i29.html</a></td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>WPI, Inc.</td>
			<td>500086</td>
			<td><a href="http://www.wpiinc.com/">www.wpiinc.com/</a></td>
		</tr>
		<tr>
			<td>Dumont tweezers #5</td>
			<td>WPI, Inc.</td>
			<td>14101</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>72000</td>
			<td><a href="http://www.emsdiasum.com/">www.emsdiasum.com</a></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Metler Toledo</td>
			<td>AB54-S/FACT</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>pH meter and electrode</td>
			<td>Beckman Coulter</td>
			<td>pHI 350</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>60129</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>63020</td>
			<td>1.0 M solution</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>21114</td>
			<td>1.0 M solution</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>431788</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Ames medium</td>
			<td>Sigma-Aldrich</td>
			<td>A1420</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>BaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>B0750</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>DL-AP4</td>
			<td>Tocris Bioscience</td>
			<td>101</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Succinic acid disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>224731</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>L-Glutamic acid</td>
			<td>Sigma-Aldrich</td>
			<td>G2834</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Leibovitz culture medium L-15</td>
			<td>Sigma-Aldrich</td>
			<td>L4386</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>MEM vitamins</td>
			<td>Sigma-Aldrich</td>
			<td>M6895</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M5550</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Airgas</td>
			<td>UN3156</td>
			<td>5% CO2</td>
		</tr>
	</tbody>
</table>

simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Bu koyu adapte yabani tip flaş yanıtları kaydedildi deney protokolleri farklı standart perfüzyon için solüsyonlar (Şekil 2) ile, yukarıda tarif edilen ve Şekil 1 &#39;de gösterilen izleyerek (WT), C57BL / 6 fare retinaları. tepki dalga ve kinetik yanı sıra çubuk fotoreseptör duyarlılığı Ames &quot;ve Locke&#39;un medya (Şekil 2A ve B) benzer çıktı. Öte yandan, HEPES-tamponlu Ringer çözeltisi (Resim bikarbonat ya da% 5 CO ...

Watch this Scientific Journal Video about Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System at JoVE.com

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Eşzamanlı<em&gt; Ex vivo</emİki retina&gt; Fonksiyonel Test tarafından<em&gt; In vivo</em&gt; Elektroretinogram Sistemi

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

simultaneous-ex-vivo-functional-testing-two-retinas-vivo

Research

JoVE Journal

Neuroscience

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

9.0K Görüntüleme.  Washington University in St. Louis. Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Video: Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Fare Retina Ganglion Hücreleri Transfeksiyon tarafından In vivo Elektroporasyon

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Nörobilim

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Ultrasonik Kan-beyin bariyeri bozulması ve Manganez kontrastlı MRG kullanma İşlevsel beyin görüntüleme

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

Polyribosome Fraksiyonu tarafından Santral Sinir Sistemi Translatome in vivo sorguluyor

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

Bozulmamış Vomeronasal Organ ve Aksesuar Koku Ampul Ex Vivo Hazırlıklar

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Eşzamanlı İki foton<em&gt; İn Vivo</em&gt; Fare Retrosplenial kortekste sinaptik Girişlerin Görüntüleme ve postsinaptik Hedefler

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Beyin Dilimleri Korku Devre Ex Vivo optogenetic Diseksiyon

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and cardiac arrest are remarkably sexually dimorphic diseases, and both induce cerebral ischemia. However, progress in understanding the vascular cognitive impairment, and then developing sex-specific treatments, has been partly limited by challenges in investigating the brain microcirculation from mouse models in functional studies. Here, we present an approach to examine the capillary-to-arteriole signaling in an ex vivo hippocampal capillary-parenchymal arteriole (HiCaPA) preparation from mouse brain. We describe how to isolate, cannulate, and pressurize the microcirculation to measure arteriolar diameter in response to capillary stimulation. We show which appropriate functional controls can be used to validate the HiCaPA preparation integrity and display typical results, including testing potassium as a neurovascular coupling agent and the effect of the recently characterized inhibitor of the Kir2 inward rectifying potassium channel family, ML133. Further, we compare the responses in preparations obtained from male and female mice. While these data reflect functional investigations, our approach can also be used in molecular biology, immunochemistry, and electrophysiology studies.

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

Fonksiyonel Çalışma için Ex Vivo Basınçlı Hipokampal Kapiller-Parenkimal Arteriyol Hazırlığı

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupiller İki Foton Fare Retinasının In vivo Görüntülenmesi

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

Ex Vivo Elektroretinogramın Küçük ve Büyük Gözlerde Retina Fonksiyonunu İncelemesi için Kurulum ve Koşulların Optimize Edilmesi

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in migraine pathophysiology has been well established, first by its potent vasodilator properties and subsequently by its presence and function as a neurotransmitter in the sensory trigeminovascular system. The migraine-provoking ability of CGRP gave support to the pharma industry to develop monoclonal antibodies and antagonists inhibiting the effect of CGRP. A new treatment paradigm has proven effective in the prophylactic treatment of migraine. One of the useful tools to further understand migraine mechanisms is the ex vivo model of CGRP release from the trigeminovascular system. It is a relatively simple method that can be used with various pharmacological tools to achieve know-how to further develop new effective migraine treatments. The present protocol describes a CGRP release model and the technique to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents. A procedure describing the experimental approach from euthanasia to the measurement of protein levels is provided. The essential isolation of the trigeminal ganglion and the trigeminal nucleus caudalis from both mice and rats and the preparation of rat dura mater are described in detail. Furthermore, representative results from both species (rats and mice) are presented. The technique is a key tool to investigate the molecular mechanisms involved in migraine pathophysiology by using various pharmacological compounds and genetically modified animals.

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

The present protocol describes the ex vivo calcitonin gene-related peptide (CGRP) release model and the strategy to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents.

Eski Vivo Kemirgenlerde Trigeminovasküler Sistemden Kalsitonin Geni ile İlgili Peptitin Salınımı

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in ...